Thymidine Synthesis Research Articles

Thymidylate Synthase Inhibition with the Novel Fluoropyrimidine FdUMP[10] Is Highly Effective Against Acute Lymphoblastic Leukemia

AbstractAbstract 1505Despite significant improvements in cure rates in pediatric patients, outcomes in adults with acute lymphoblastic leukemia (ALL) remain dismal with an estimated 5-year survival of less than 10% for patients over the age of 60. Compared to children, ALL in adults is characterized by an increased incidence of poor prognostic cytogenetics, lower complete remission rates, and higher relapse rates. Once relapse occurs in adults, the outcome is truly dismal, with a 6% salvage rate with current therapies. Initial treatment of ALL combines essentially all currently used agents, leaving very few therapies once relapse occurs. There is a clear need for better therapeutic options for these patients.5-Fluorouracil (5-FU) is a fluoropyrimidine (FP) prodrug that must first be converted to 5-fluoro-2’-deoxyuridine-5’-O-monophosphate (FdUMP) in order to inhibit thymidylate synthase (TS) and shut down tumor thymidine synthesis. It can also be metabolized to a ribose metabolite that interferes with RNA processing and leads to toxicity in any transcriptionally active cell. The profound GI toxicities of 5-FU and lack of potency against ALL has lead to the abandonment of TS inhibition in the treatment of ALL. In contrast, FdUMP[10] is a oligodeoxynucleotide containing 10 FdUMP moieties linked by phosphodiester bonds. FdUMP[10] has profound activity against preclinical models of acute myeloid leukemia (AML), with minimal toxicities. The human T cell ALL cell line, Jurkat, was extremely sensitive to FdUMP[10], with an IC50of 5.4nM (95% CI 4.609–6.417). We sought to determine the activity of FdUMP[10] against additional preclinical ALL models.In vitro, FdUMP[10] exhibited remarkable activity against the human T and B Cell ALL cell lines, DG75, Molt-4, CCRF-CEM, and SUP-B15 with an average IC50 value of 1.83nM (Range 0.21–4.1nM). In two murine ALL cell lines driven by expression of BCR-ABL, FdUMP[10] exhibited even greater activity, with an average IC50 value of 0.662nM (range 0.125–1.2nM). For comparison we also tested 5-FU and cytarabine in the same assay against DG75, MOLT-4 and a BCR-ABL driven murine line. In all cases FdUMP[10] was the most potent agent and was more than 1000 times more potent than 5-FU despite having only 10 times the FP content. In vivo, FdUMP[10] treatment provided a statistically significant increase in survival in a BCR-ABL driven, syngeneic ALL mouse model (p=0.0002 by log rank test). In a separately derived syngeneic model expressing the T315I variant of BCR-ABL, treatment with FdUMP[10] significantly prolonged survival (p=0.0013).In AML models exposure to FdUMP[10] resulted in apoptosis. To assess if ALL cells undergo a similar apoptotic response we exposed human and mouse ALL cells to FdUMP[10] and assessed for apoptosis induction via propidium iodide (PI) and Annexin V staining. As we found previously in AML models, FdUMP[10] exposure resulted in a robust induction of Annexin V and PI staining consistent with an apoptotic response. The level of apoptosis with FdUMP[10] could not be reproduced by 5-FU even when used at 100 times higher concentration.To determine the effect of FdUMP[10] on thymidylate synthase (TS) activity in ALL, Jurkat cells were exposed to 10nM FdUMP[10] or 100nM 5- FU and TS activity was assayed. We found a profound and prolonged inhibition with FdUMP[10] compared to 5-FU, despite the identical amount of fluoropyrimidine.FdUMP[10] is synergistic with doxorubicin and cytarabine in AML models. To assess whether FdUMP[10] could synergize with targeted therapy in ALL, Baf-3 cells expressing BCR-ABL were exposed to the tyrosine kinase inhibitor nilotinib plus FdUMP[10]. The combination was synergistic, with combinatory index values of 0.7113 to 0.4803. In preliminary data in vivo, the combination of FdUMP[10] and nilotinib resulted in a greater reduction in leukemic burden then nilotinib alone.In summary, FdUMP[10] exhibited remarkable activity against human and murine ALL cells in vitro and in vivo by inducing apoptosis and profound TS inhibition, and showing a synergistic benefit when combined with nilotinib. These data demonstrate that TS is a valid target in ALL cells. This fact combined with previous studies demonstrating a favorable toxicity profile, make FdUMP[10] a promising candidate for treatment of ALL. Disclosures:Pardee:Salzburg Therapuetics: Membership on an entity's Board of Directors or advisory committees. Gmeiner:Salzburg Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Cerebral folate deficiency--mishaps and misdirection

Folic acid derivatives are important biological compounds that occupy key roles in a variety of cellular metabolic pathways including the synthesis of purines and thymidine, the remethylation of homocysteine to form methionine and the synthesis and breakdown of a number of amino acids, neurotransmitters and myelin (Watkins and Rosenblatt, 2012). Folates are critical for foetal development, and folate deficiency has been associated with an increased risk of congenital and post-natal problems including neural tube, cardiovascular and limb defects; cleft lip and palate; chromosomal disorders, such as Trisomy 21 and Fragile X and some paediatric malignancies including leukaemia and neuroblastoma. Following a report by the Medical Research Council Vitamin Study Research Group (1992), maternal dietary supplementation with folic acid has been promoted in many countries, and the current UK recommendation (in accordance with WHO guidance) is for 400 µg/day of folic acid, commencing preconception and continuing up to 12 weeks gestation (NICE, 2008). Structurally, the biologically active folates consist of a pteridine moiety linked to a variable number of glutamate molecules. Polyglutamated folates do not cross lipid membranes easily and require deconjugation to a monoglutamate before traversing the cell and mitochondrial membranes. Folate is primarily transported as 5-methyltetrahydrofolate (5-MTHF), and this is the major form of folate found in both plasma and CSF (Hyland et al. , 2010). 5-MTHF reaches the CNS largely through choroid plexus epithelial cells, where transport across the cell membrane is achieved predominantly by a high-affinity, low-capacity endocytic process involving …

Abstract 376: Limits of [18F]-FLT PET as a predictive imaging biomarker of proliferation in oncology

Abstract Background: Surrogate, non-invasive imaging biomarkers of cellular proliferation hold great promise for quantifying response to personalized medicine in oncology. An emerging imaging approach to assess tumor proliferation utilizes the positron emission tomography (PET) tracer 3′-deoxy-3′[18F]-fluorothymidine, [18F]-FLT. Though several studies have associated serial changes in [18F]-FLT-PET with elements of therapeutic response, the degree to which [18F]-FLT uptake quantitatively reflects proliferative index has been continuously debated for more that a decade. The goal of this preclinical study was to elucidate quantitative relationships between [18F]-FLT-PET and cellular, proteomic, and genomic metrics proliferation in treatment naive tumors. Methods and Findings: [18F]-FLT-PET imaging was conducted in mice bearing human colorectal or breast cancer cell line xenografts. Quantitative relationships between PET imaging, thymidine kinase 1 (TK1) protein and RNA levels, and immunostaining for proliferation markers (Ki67, TK1) were evaluated using imaging-matched tumor specimens. Overall, we found that [18F]-FLT-PET reflects TK1 protein levels in tumors. However, the cell cycle specificity of TK1 expression and the variable degree to which tumors utilize thymidine salvage for DNA synthesis tend to decouple [18F]-FLT-PET data from estimates of proliferation by standard, clinically employed means. Conclusions: Our findings illustrate that [18F]-FLT-PET reflects tumor proliferation as a function of thymidine salvage pathway utilization. Therefore, unlike more generalizable proliferation markers, such as Ki67 immunostaining, tumor uptake of [18F]-FLT reflects proliferative indices to variable and potentially unreliable extents. [18F]-FLT-PET can not discriminate moderately proliferative, thymidine salvage driven disease from highly proliferative tumors that rely primarily upon de novo thymidine synthesis. For those reasons, the magnitude of [18F]-FLT uptake in treatment naive tumors should not be considered a surrogate of proliferative index. These data provide rationale for the diversity of correlative results reported, both positive and discordant, comparing [18F]-FLT-PET and tumor proliferation across numerous single-center clinical studies and provide a basis for future clinical best-practices when this imaging biomarker is employed in oncology. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 376. doi:1538-7445.AM2012-376

Teratogen update: Methotrexate

Methotrexate and aminopterin are folic acid antagonists that inhibit dihydrofolate reductase, resulting in a block in the synthesis of thymidine and inhibition of DNA synthesis. Methotrexate has been used for the treatment of malignancy, rheumatic disorders, and psoriasis and termination of intrauterine pregnancy. Recently, methotrexate has become a standard treatment for ectopic pregnancy. The misdiagnosis of an intrauterine pregnancy as an ectopic pregnancy can result in exposure of a continuing pregnancy to dose levels of methotrexate of 50 mg/m(2) (maternal body surface area). Experimental animal studies have associated methotrexate therapy with embryo death in mice, rats, rabbits, and monkeys. Structural malformations have been most consistently produced in rabbits at a maternal dose level of 19.2 mg/kg. Abnormalities in rabbits include hydrocephalus, microphthalmia, cleft lip and palate, micrognathia, dysplastic sacral and caudal vertebrate, phocomelia, hemimelia, syndactyly, and ectrodactyly. Based on human case reports of methotrexate exposure during pregnancy, a methotrexate embryopathy has been described that includes growth deficiency, microcephaly, hypoplasia of skull bones, wide fontanels, coronal or lambdoidal craniosynostosis, upswept frontal scalp hair, broad nasal bridge, shallow supraorbital ridges, prominent eyes, low-set ears, maxillary hypoplasia, epicanthal folds, short limbs, talipes, hypodactyly, and syndactyly. This syndrome may be associated with exposures between 6 and 8 weeks after conception and dose levels of 10 mg/week or greater. More recent case reports of methotrexate exposure for the misdiagnosis of ectopic pregnancy involve treatment before 6 weeks after conception and have raised the suggestion of a distinct syndrome due to such early exposures. Tetralogy of Fallot and perhaps other neural crest cell-related abnormalities may be features of this early syndrome. A disproportionality analysis of methotrexate and aminopterin case reports and series provides support for pulmonary atresia, craniosynostosis, and limb deficiencies as reported more often than expected in methotrexate-exposed children. Denominator-based data will be welcome to better define elements of a methotrexate embryopathy and possibly to distinguish an early exposure syndrome from anomalies traditionally associated with methotrexate exposure.

The gene encoding the alternative thymidylate synthase ThyX is regulated by sigma factor SigB in Corynebacterium glutamicum ATCC 13032

Both ThyA and ThyX proteins catalyze the transfer of the methyl group from methylenetetrahydrofolate (CH(2) H(4) -folate) to dUMP, forming dTMP. To estimate the relative steady state expression levels of ThyA and ThyX, Western blot analysis was performed using ThyA or ThyX antiserum on total protein from the wild-type, ΔthyX, and thyX-complemented strains of Corynebacterium glutamicum. The level of ThyA decreased gradually during the stationary growth phase but that of ThyX was maintained steadily. Whereas the expression level of ThyA in a ΔsigB strain was comparable to that of the wild-type, the level of ThyX was significantly diminished in the deletion mutant and was restored to that of the wild-type in the complemented strain, indicating that the level of ThyX was regulated by SigB. Growth of the C.glutamicum ΔsigB strain was dependent upon coupling activity of dihydrofolate reductase (DHFR) with ThyA for the synthesis of thymidine, and thus showed sensitivity to the inhibition of DHFR by the experimental inhibitor, WR99210-HCl. These results suggested that the relative levels of ThyA and ThyX differ in response to different growth phases and that SigB is necessary for maintenance of the level of ThyX during transition into the stationary growth phase.

Amnionless Expression in the Mouse Testis and Leydig Cells and Its Change in Leydig Cell Hyperplasia.

In testis, vitamin B12 (cobalmin)-deficiency resulted in atrophy of seminiferous tubules and aplasia of sperms and spermatids. Deficiency in vitamin B12 decreased in methionine synthase activity. Vitamin B12 involves in DNA synthesis as it participates in methionine synthase followed by thymidine synthesis. Amnionless (Amn), a transmembrane protein directs endocytosis of cubilin with its ligand, contributing intrinsic factor-vitamin B12 absorption. Expressional profile from data base revealed that Amn is considerably expressed in kidney, small intestine, placenta, and testis in mouse. In an effort to understand vitamin B12 transport to the testicular cells, we analyzed the Amn gene expression in the developing mouse testis and Leydig cells. In the meantime, change of Amn expression in Leydig cell hyperplasia by flutamide an antiandrogen was examined in testis. In mouse testis, Amn mRNA levels were low until 14 days post partum (pp), markedly increased at puberty onwards. In the interstitium, Amn mRNA levels were low until 14 days pp, markedly increased together with 3β-hydroxysteroid dehydrogenase type VI mRNA at puberty (28 days pp), and further increased at 56 days pp. AMN immunoreactivity was found weakly in Sertoli cells throughout the testis development. Early spermatocytes showed strong AMN immunoreactivity at 14 days pp onwards, suggesting that AMN participates in the early meiosis. In Leydig cells, AMN immunoreactivity was not found until 14 days pp but strongly expressed at 28 days pp onwards. Quantitative image analysis revealed that AMN immunoreactivity in Leydig cells was increased by 2.8 fold between 28 and 56 days pp. Mean size of AMN-positive Leydig cells was increased by 1.8 fold between 14 and 28 days pp and 1.5 fold between 28 and 56 days pp. This suggests positive relationship between AMN expression and functional differentiation of adult Leydig cells. In adult male mice, oral administration of flutamide (100 mg/kg/day) for 10 days induced a Leydig cell hyperplasia in mouse testis in which Amn mRNA and AMN immunoreactivity were increased in Leydig cells, suggesting that AMN may participate in the pathogenesis of Leydig cell hyperplasia. In adult Leydig cells primary culture, flutamide (100 nM) increased Amn mRNA expression, suggesting that Amn gene expression is negatively coupled with androgen receptor activation. In conclusion, AMN may participate in the meiosis of early spermatocytes and functional differentiation of adult Leydig cells via mediating the vitamin B12 transport in mouse testis. In addition, AMN may participate in the Leydig cell hyperplasia via promoting cell proliferation in Leydig cell via stimulating DNA synthesis. This is the first report on the AMN expression in male germ cells and somatic cells in mammalian testis. (poster)

Histone deacetylase inhibitors induce thymidine phosphorylase expression in cultured breast cancer cell lines

Thymidine phosphorylase (TP) is an enzyme involved in thymidine synthesis and degradation. The expression of this enzyme has been proposed as a predictive factor for the therapeutic benefit of capecitabine, which is a precursor of the drug 5'-fluorouracil. In fact, TP is the rate-limiting enzyme in the activation of capecitabine. Therefore, higher levels of TP are expected to sensitize cancer cells to capecitabine treatment. In the present study, using breast cancer cell lines, we found a correlation between TP mRNA and protein levels, suggesting that compounds able to increase TP gene expression also increase protein levels. Accordingly, we demonstrated that treatment of breast cancer MCF7 and MDA231 cell lines with histone deacetylase inhibitors, tricostatin A and suberoylanilide hydroxamic acid, increased TP both at the mRNA and protein level. The effects of histone deacetylase inhibitors were not found to occur via the cytokine TNFα, a known inducer of TP expression. Our findings suggest a strategy to sensitize breast cancer cells to capecitabine treatment.

Imaging Colon Cancer Response Following Treatment with AZD1152: A Preclinical Analysis of [18F]Fluoro-2-deoxyglucose and 3′-deoxy-3′-[18F]Fluorothymidine Imaging

To determine whether treatment response to the Aurora B kinase inhibitor, AZD1152, could be monitored early in the course of therapy by noninvasive [(18)F]-labeled fluoro-2-deoxyglucose, [(18)F]FDG, and/or 3'-deoxy-3'-[(18)F]fluorothymidine, [(18)F]FLT, PET imaging. AZD1152-treated and control HCT116 and SW620 xenograft-bearing animals were monitored for tumor size and by [(18)F]FDG, and [(18)F]FLT PET imaging. Additional studies assessed the endogenous and exogenous contributions of thymidine synthesis in the two cell lines. Both xenografts showed a significant volume-reduction to AZD1152. In contrast, [(18)F]FDG uptake did not demonstrate a treatment response. [(18)F]FLT uptake decreased to less than 20% of control values in AZD1152-treated HCT116 xenografts, whereas [(18)F]FLT uptake was near background levels in both treated and untreated SW620 xenografts. The EC(50) for AZD1152-HQPA was approximately 10 nmol/L in both SW620 and HCT116 cells; in contrast, SW620 cells were much more sensitive to methotrexate (MTX) and 5-Fluorouracil (5FU) than HCT116 cells. Immunoblot analysis demonstrated marginally lower expression of thymidine kinase in SW620 compared with HCT116 cells. The aforementioned results suggest that SW620 xenografts have a higher dependency on the de novo pathway of thymidine utilization than HCT116 xenografts. AZD1152 treatment showed antitumor efficacy in both colon cancer xenografts. Although [(18)F]FDG PET was inadequate in monitoring treatment response, [(18)F]FLT PET was very effective in monitoring response in HCT116 xenografts, but not in SW620 xenografts. These observations suggest that de novo thymidine synthesis could be a limitation and confounding factor for [(18)F]FLT PET imaging and quantification of tumor proliferation, and this may apply to some clinical studies as well.

Analysis of Coevolving Gene Families Using Mutually Exclusive Orthologous Modules

Coevolutionary networks can encapsulate information about the dynamics of presence and absence of gene families in organisms. Analysis of such networks should reveal fundamental principles underlying the evolution of cellular systems and the functionality of sets of genes. In this study, we describe a new approach for analyzing coevolutionary networks. Our method detects Mutually Exclusive Orthologous Modules (MEOMs). A MEOM is composed of two sets of gene families, each including gene families that tend to appear in the same organisms, such that the two sets tend to mutually exclude each other (if one set appears in a certain organism the second set does not). Thus, a MEOM reflects the evolutionary replacement of one set of genes by another due to reasons such as lineage/environmental specificity, incompatibility, or functional redundancy. We use our method to analyze a coevolutionary network that is based on 383 microorganisms from the three domains of life. As we demonstrate, our method is useful for detecting meaningful evolutionary clades of organisms as well as sets of proteins that interact with each other. Among our results, we report that: 1) MEOMs tend to include gene families whose cellular functions involve transport, energy production, metabolism, and translation, suggesting that changes in the metabolic environments that require adaptation to new sources of energy are central triggers of complex/pathway replacement in evolution. 2) Many MEOMs are related to outer membrane proteins, such proteins are involved in interaction with the environment and could thus be replaced as a result of adaptation. 3) MEOMs tend to separate organisms with large phylogenetic distance but they also separate organisms that live in different ecological niches. 4) Strikingly, although many MEOMs can be identified, there are much fewer cases where the two cliques in the MEOM completely mutually exclude each other, demonstrating the flexibility of protein evolution. 5) CO dehydrogenase and thymidylate synthase and the glycine cleavage genes mutually exclude each other in archaea; this may represent an alternative route for generation of methyl donors for thymidine synthesis.

Thymidylate synthase gene polymorphisms and markers of DNA methylation capacity

Background DNA methylation plays a critical role in gene regulation and has been implicated in the etiology of chronic disease including atherosclerosis, neural degeneration and cancer. One-carbon metabolism serves two critically important functions: one concerning the production of purines and thymidine for DNA synthesis and the other related to the provision of methyl groups through the metabolism of methionine. Critical intermediates of methionine metabolism relevant to DNA methylation include S-adenosylmethionine (SAM), a universal methyl donor, and S-adenosylhomocysteine (SAH), a potent inhibitor of most methylation reactions. Thymidine synthesis, catalyzed by the crucial enzyme thymidylate synthase (TS), competes with methionine metabolism for a common substrate. Three functional polymorphisms in the TS gene have been identified including: (i) the thymidylate synthase enhancer region (TSER) tandem repeat polymorphism and (ii) the G to C single nucleotide polymorphism (G/C SNP) both of which occur in the 5′untranslated region (UTR) of the TS gene; and (iii) the 6-bp deletion at base pair 1494 (TS1494del6) located in the 3′UTR. Purpose The purpose of this research was to investigate the relationship between TS polymorphisms and concentrations of SAM and SAH, markers of DNA methylation capacity. Methods The study population consisted of 395 healthy male and female volunteers from Kingston, Ontario and Halifax, Nova Scotia, Canada between 2006 and 2008. The effect of each TS polymorphism on SAM and SAH concentrations was investigated, and further analyses were conducted on categorization of polymorphisms based on 5′ or 3′UTR. The combined effect of TS polymorphisms on SAM and SAH concentrations was also investigated, in addition to interactions between polymorphisms in TS and MTHFR 677C>T and interactions between TS polymorphisms and serum folate and vitamin B 12 status. Results No associations were observed between TS polymorphisms and concentrations of SAM and SAH. Analysis of interaction between TS and MTHFR polymorphisms on SAH levels revealed a significant interaction with TS 3′polymorphism and MTHFR C677T ( p = 0.03). As well, interactions between TS 3′polymorphism and serum folate ( p = 0.03) and the combined effect of TS polymorphisms and serum folate on SAH levels ( p = 0.04) were found. Conclusions The findings of this research provide evidence that SAH, a marker of methylation capacity, is influenced by genetic and environmental factors and their interactions.

Thymidylate Synthase (TS) Expression In Diffuse Large B-Cell Lymphoma (DLBCL): Identification of a Potential Therapeutic Target

AbstractAbstract 5107 Purpose:TS is critical for the de novo synthesis of thymidine needed for tumor cell proliferation and DNA damage repair, whereas thymidine kinase 1 (TK1) is key to the salvage pathway providing thymidine recovered from plasma. While TK1 expression has been previously shown to correlate with proliferation in DLBCL, little is known about TS levels in these neoplasms. We hypothesized that, similarly to TK1, TS expression levels associate with increased proliferation and thus may represent a novel therapeutic target in large cell lymphoma. Methods:To address this hypothesis, we determined the expression of TS in lymph node biopsies from 43 DLBCL patients sequentially enrolled in the study with no pre-selection criteria other than the primary histological diagnosis of lymphoma, and then compared it to TK1 expression. In addition, Ki-67 (MIB1) was included as an established marker of cell proliferation. Pearson correlation along with the correlation coefficient and two-tailed P values were calculated to measure the extent of covariation. Results:TS expression and both MIB1 and TK1 were significantly correlated with correlation coefficients of 0.43 and 0.65, respectively (95% CI 0.13 to 0.65 and 0.44 to 0.8; P values = 0.006 and <0.0001, respectively). The correlation between TS and MIB1 expression was in fact stronger than between MIB1 and TK1 (P values 0.006 and 0.04, respectively). Mean percent positive cells and standard deviations for MIB1, TS and TK1 were 44.5% (SD=35), 51.8% (SD=33.4), and 44% (SD=28.5), respectively. Despite the general good correlation, however, six patients had relatively high TS levels (defined as % cell staining above the mean for TS) and relatively low TK1 levels (% cell staining below the mean for TK1), while the reverse was true in seven patients. Eight patients had high levels of TK1 and TS with ≥ 70% cells positive for both; seven patients had low levels of both proteins (≤30% of cells stained). Conclusion and Significance:This is the first report of TS expression in DLBCL. TS is frequently expressed in DLBCL and there is a significant correlation between TS and TK1 or MIB1 expression. Further studies are warranted to determine the correlation between TS or TK1 expression and patient prognosis in comparison to MIB1. Importantly, the common expression of TS in patients with DLBCL indicates that TS may be a suitable target for TS inhibitors (TSIs) in these neoplasms. Preclinical studies are underway to test this hypothesis. Disclosures:No relevant conflicts of interest to declare.

ChemInform Abstract: Synthesis of Thymidine from 5-Iodo-2′-deoxyuridine.

ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

ChemInform Abstract: Synthesis and Substrate Properties of Thymidine 5′-Triphosphate Analogues with Large Hydrophobic Substituent Groups at α-P Atom.

ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Identification of thymidylate synthase as a potential therapeutic target for lung cancer

Background:Thymidylate synthase (TS), a key enzyme in the de novo synthesis of thymidine, is an important chemotherapeutic target for malignant tumours including lung cancer. Although inhibition of TS has an antiproliferative effect in cancer cells, the precise mechanism of this effect has remained unclear.Methods:We examined the effects of TS inhibition with an RNA interference-based approach. The effect of TS depletion on the growth of lung cancer cells was examined using colorimetric assay and flow cytometry.Results:Measurement of the enzymatic activity of TS in 30 human lung cancer cell lines revealed that such activity differs among tumour histotypes. Almost complete elimination of TS activity by RNA interference resulted in inhibition of cell proliferation in all tested cell lines, suggestive of a pivotal role for TS in cell proliferation independent of the original level of enzyme activity. The antiproliferative effect of TS depletion was accompanied by arrest of cells in S phase of the cell cycle and the induction of caspase-dependent apoptosis as well as by changes in the expression levels of cyclin E and c-Myc. Moreover, TS depletion induced downregulation of the antiapoptotic protein X-linked inhibitor of apoptosis (XIAP), and it seemed to activate the mitochondrial pathway of apoptosis.Conclusion:Our data provide insight into the biological relevance of TS as well as a basis for clinical development of TS-targeted therapy for lung cancer.

Increase of [18F]FLT Tumor Uptake In Vivo Mediated by FdUrd: Toward Improving Cell Proliferation Positron Emission Tomography

3'-deoxy-3'-[(18)F]fluorothymidine ([(18)F]FLT), a cell proliferation positron emission tomography (PET) tracer, has been shown in numerous tumors to be more specific than 2-deoxy-2-[(18)F]fluoro-D-glucose ([(18)F]FDG) but less sensitive. We studied the capacity of a nontoxic concentration of 5-fluoro-2'-deoxyuridine (FdUrd), a thymidine synthesis inhibitor, to increase uptake of [(18)F]FLT in tumor xenografts. The duration of the FdUrd effect in vivo on tumor cell cycling and thymidine analogue uptake was studied by varying FdUrd pretreatment timing and holding constant the timing of subsequent flow cytometry and 5-[(125)I]iodo-2'-deoxyuridine biodistribution measurements. In [(18)F]FLT studies, FdUrd pretreatment was generally performed 1 h before radiotracer injection. [(18)F]FLT biodistributions were measured 1 to 3 h after radiotracer injection of mice grafted with five different human tumors and pretreated or not with FdUrd and compared with [(18)F]FDG tumor uptake. Using microPET, the dynamic distribution of [(18)F]FLT was followed for 1.5 h in FdUrd pretreated mice. High-field T2-weighted magnetic resonance imaging (MRI) and histology were used comparatively in assessing tumor viability and proliferation. FdUrd induced an immediate increase in tumor uptake of 5-[(125)I]iodo-2'-deoxyuridine, that vanished after 6 h, as also confirmed by flow cytometry. Biodistribution measurements showed that FdUrd pretreatment increased [(18)F]FLT uptake in all tumors by factors of 3.2 to 7.8 compared with controls, while [(18)F]FDG tumor uptake was about fourfold and sixfold lower in breast cancers and lymphoma. Dynamic PET in FdUrd pretreated mice showed that [(18)F]FLT uptake in all tumors increased steadily up to 1.5 h. MRI showed a well-vascularized homogenous lymphoma with high [(18)F]FLT uptake, while in breast cancer, a central necrosis shown by MRI was inactive in PET, consistent with the histomorphological analysis. We showed a reliable and significant uptake increase of [(18)F]FLT in different tumor xenografts after low-dose FdUrd pretreatment. These results show promise for a clinical application of FdUrd aimed at increasing the sensitivity of [(18)F]FLT PET.

Alternative thymidylate synthase, ThyX, involved in Corynebacterium glutamicum ATCC 13032 survival during stationary growth phase

A BLASTP search has shown the presence of a gene homologous to an alternative thymidylate synthase (TS), thyX, in Corynebacterium glutamicum ATCC 13032. To determine if thyX is functionally analogous to thyA, thyX was cloned in a plasmid and the resulting construct was transferred by transformation into a thyA mutant of Escherichia coli. The ThyX from C. glutamicum compensated for the defect in TS-deficient E. coli. A functional knockout of the thyX gene was constructed by allelic replacement using a sucrose counter-selectable suicide plasmid and confirmed by PCR and reverse transcriptase-PCR analyses. This mutant was viable without thymidine supplementation, suggesting that thyX is not an essential gene in C. glutamicum. Growth of the thyX mutant was dependent upon coupling activity of dihydrofolate reductase (DHFR) with ThyA for the synthesis of thymidine, and thus showed sensitivity to the inhibition of DHFR by the experimental inhibitor, WR99210. This indicates that thymidine synthesis was at least partially dependent on thyX expression. As it approached stationary phase, the thyX mutant lost viability much more rapidly than the parental wild type and the mutant complemented the thyX gene, suggesting that the activity of the ThyX enzyme is important in that phase of the growth cycle.

Iron status and serine hydroxymethyltransferase (SHMT) activity and abundance in pre‐ and postmenopausal women

To explore relationships between iron status and lymphocyte SHMT activity (ACT) and abundance (ABN), 118 postmenopausal women (54.3 y) enrolled in SIRBL (Soy Isoflavones for Reducing Bone Loss) study and 105 young women (21.2 y) were studied. Serum ferritin, folate, and vitamin B‐12, plasma total homocysteine (tHcy) and pyridoxal phosphate (PLP), lymphocyte SHMT ACT and ABN were determined. Subjects in the first (4.8 ng/mL; 93% premenopausal) and fifth (92.6 ng/mL; 98% postmenopausal) quintiles of serum ferritin were compared. The fifth quintile had higher median BMI (25 vs 22 kg/m2; p&lt;0.02), plasma PLP (p&lt;0.0001), total homocysteine (p&lt;0.05), and SHMT ABN (p&lt;0.0001), but significantly lower SHMT ACT (p&lt;0.02) than first quintile. Ferritin quintile Folate ng/mL B‐12 pg/mL PLP nmol/L tHcy nmol/L SHMT ACT cpm/ug protein SHMT ABN relative density First (n) 14.2 (45) 597 (45) 61.7 (45) 6.4 (45) 43.6 (27) 1.1 (21) Fifth (n) 13.1 (45) 521 (45) 93.9 (45) 6.6 (45) 13.0 (14) 12.3 (31) Increased iron status may enhance SHMT expression, favoring thymidine synthesis over Hcy remethylation, thus warranting further investigation.Funded by USDA Special Grant, NWRC, Iowa State University.

Toxicité sévère à la capécitabine liée à un déficit enzymatique en dihydropyrimidine déshydrogénase (DPD)

Capecitabine is an anticancer agent, prodrug of 5 fluorouracil (5-FU) administered orally and with a narrow therapeutic index. In gastrointestinal cancer, capecitabine is indicated for the treatment of colorectal cancer and metastatic unresectable gastric cancer. The 5-FU is active by incorporation in the biosynthesis of nucleic acids. Inhibition of endogenous synthesis of thymidine is the main way of toxicity of 5-FU. 5-FU is metabolised by the dihydopyrimydine dehydrogenase (DPD). Patients with a DPD deficiency can experience severe toxicity of 5-FU. We report the case of a patient who presented signs of major toxicity justifying hospitalization in intensive care unit 11 days after capecitabine initiation. Investigations showed that he had a DPD deficiency. This case leads to explain the different biological ways to identify patients at risk of developing severe toxicity following capecitabine administration because of DPD deficiency. Is it possible to make a systematic screening before initiation of treatment with 5-FU or prodrug of 5-FU?

Synthesis of Carbon E,E-Diene Chain-Linked Dinucleotide Analogues

The synthesis of a dinucleotide thymidine―thymidine linked by a carbon E,E-diene chain is described. This dimer is synthesized by a coupling reaction between an (E)-vinylstannane and an (E)-iodovinyl partner prepared from acetylenic parents, which are both available from thymidine in six and five steps, respectively. In addition, a new efficient access to 3'-C-formyl thymidine is presented.

Functionalized thymidine derivatives as carriers for the γ-emitter technetium tricarbonyl moiety

Nuclear medicine imaging of cell proliferation has gained broad interest in clinical oncology. A good tracer to image cell proliferation, possibly associated to tumour progression, should rapidly and specifically be incorporated into growing DNA. Following this idea, we have singled out thymidine as a potential biological carrier for delivery of the organometallic fragment [M(CO) 3] + (M = “cold” Re, γ-emitter 99mTc) to DNA. In the present paper we report the two-step synthesis of thymidine N-3 derivatives and their coordination complexes. Thymidine could be alkylated without protecting the sugar alcoholic functions. Methyl-2,2′-diaminodiethylamine thymidine derivative was quickly reacted with rhenium and technetium carbonyls in almost quantitative yield. The resulting 99mTc complex represents a convenient radiopharmaceutical for single photon emission tomography (SPECT).